Diptera: Tephritidae - BioOne

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INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Toxicities and Synergistic Effects of Several Insecticides Against the Oriental Fruit Fly (Diptera: Tephritidae) JING-JING WANG, DONG WEI, WEI DOU, FEI HU, WEI-FENG LIU,

AND

JIN-JUN WANG1

Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400716, P. R. China

J. Econ. Entomol. 106(2): 970Ð978 (2013); DOI: http://dx.doi.org/10.1603/EC12434

ABSTRACT The oriental fruit ßy, Bactrocera dorsalis (Hendel), is a serious insect pest that causes large losses to orchards globally. In this study, we conducted experiments to investigate the susceptibility of two populations (Kunming of Yunnan and Dongguan of Guangdong province) of B. dorsalis to nine insecticides. Bioassay results demonstrated that Þpronil was the most effective insecticide, followed by phoxim, abamectin, triazophos, ␤-cypermethrin, chlorpyrifos, deltamethrin, malathion, and imidacloprid against the Kunming of Yunnan province population, with LD50 values that ranged from 1.55 to 187.48 ng/ßy. For the Dongguan of Guangdong province population, Þpronil was also most toxic, followed by triazophos, phoxim, chlorpyrifos, abamectin, deltamethrin, ␤-cypermethrin, malathion, and imidacloprid, with LD50 values from 2.07 to 439.11 ng/ßy. The addition of synergists triphenyl phosphate, piperonyl butoxide, and diethyl maleate yielded different levels of synergistic effects on different insecticides against each population. However, the synergistic effects on the nine insecticides against the two populations are different. The treatment of a sublethal dose (LD20) of ␤-cypermethrin together with three synergists could induce increased speciÞc activity of carboxylesterases at the beginning of exposure, followed by a decline within 24 h. The speciÞc activity of carboxylesterases was higher in the fat body, midgut, and Malpighian tubules, suggesting these are important tissues for detoxiÞcation. Overall, the data developed in this study provide useful information for designing an insecticide management strategy for controlling this insect in the Þeld. KEY WORDS Bactrocera dorsalis, toxicity, synergist, carboxylesterases

The oriental fruit ßy, Bactrocera dorsalis (Hendel), is one of the most economically important fruit ßy pests in East Asia and the PaciÞc (Drew and Hancock 1994, Clarke et al. 2005). The oriental fruit ßy is a polyphagous pest that damages ⬎250 plant species including various fruits and vegetables, which range widely from tropical to temperate zones (Chen and Ye 2007). Many control measures have been applied to manage this species, but the control of this pest has largely relied on chemical insecticides. In recent years, Þeld monitoring of the oriental fruit ßy has indicated that unreasonable use of chemical insecticides has resulted in serious resistance problems (Hsu et al. 2004, 2008). As a result, many Þeld populations of these species have developed resistances to multiple insecticides. For example, it has been reported that compared with a susceptible colony, the Þeld populations exhibited 4.7-fold to 594-fold resistance to different classes of insecticides including organophosphate, pyrethroid, and carbamate (Hsu et al. 2004). Resistance of this species in mainland China has varied from moderate to high level to trichlorfon, ␤-cypermethrin, and aver1 Correspondingauthor,e-mail:[email protected],wangjinjun@ swu.edu.cn.

mectin, and the highest resistance ratio was 33-fold (Zhang et al. 2007). The current resistance monitoring of B. dorsalis in several different populations from mainland China has identiÞed higher levels of resistance to many insecticides (Jin et al. 2011). Accurate detection of resistance levels to different classes of insecticides within B. dorsalis populations is important for the guidance of future management. A possible strategy to improve oriental fruit ßy control in conventional and organic orchards may be disruption of detoxiÞcation enzymes responsible for insecticide metabolism. A synergist is deÞned as a class of chemicals that has no biological activity itself and has low animal toxicity, but when mixed with pesticide compounds can inhibit speciÞc metabolic pathways, decrease the threshold of pesticide use, and thus signiÞcantly increase pesticide toxicity and efÞcacy (Young et al. 2005). Such synergized pesticides and pesticide mixture formulations have been adopted to overcome pesticide resistance in several arthropod pest populations (Ozaki 1983, Raffa and Priester 1985, Gunning et al. 1998). The most commonly used synergists include triphenyl phosphate (TPP), diethyl maleate (DEM), and piperonyl butoxide (PBO), which are normally considered as inhibitors of es-

0022-0493/13/0970Ð0978$04.00/0 䉷 2013 Entomological Society of America

April 2013

WANG ET AL.: SUSCEPTIBILITY OF B. dorsalis TO INSECTICIDES

terases, glutathione S-transferase, and cytochrome P450 monooxygenases, respectively. Through synergist experiments, we can obtain preliminary evidence of the relationship between insecticide resistance and detoxiÞcation pathways. A number of studies have indicated that by the use of synergists TPP, DEM, and PBO one could suggest that enhanced enzymatic activity is a resistance mechanism in moderately resistant populations (Ribeiro et al. 2003, Wu et al. 2004, Huang and Han 2007, Qian et al. 2008, Pang et al. 2012). Among these detoxiÞcation enzymes, carboxylesterases (CarEs) play an important role in detoxifying many endogenous and exogenous compounds, particularly pyrethroid, carbamate, and organophosphorus insecticides (Casida and Quistad 2004, Latif et al. 2010). Although CarEs are widely distributed in insect tissues, the expression and activity of CarEs in different tissues, such as fat body, midgut, and Malpighian tubules, depends strongly on the organism and growth stages (Felton and Duffey 1991, Rashad 2008, Yu et al. 2009, Zhang et al. 2011). To date, little is known about the effects of insecticide to CarEs exposure in the presence of synergist chemicals in B. dorsalis. The main objective of this study was to determine the susceptibilities of two populations of B. dorsalis to four organophosphorus insecticides (malathion, chlorpyrifos, phoxim, and triazophos), two pyrethroid insecticides (alphamethrin and deltamethrin), one bioinsecticide (abamectin), one neonicotinoid insecticide (imidacloprid), and one phenyl pyrazole insecticide (Þpronil). Our second objective was to evaluate the synergistic effect of nine insecticides with TPP, PBO, and DEM. Moreover, we investigated the possible involvement of CarEs in relation to the detoxiÞcation of ␤-cypermethrin in B. dorsalis. Materials and Methods Insects. Individuals of B. dorsalis were collected from Kunming of Yunnan province (KM population) and Dongguan of Guangdong province (DG population) in China in 2008. To obtain the required number of individuals, the newly hatched larvae were fed an artiÞcial diet consisting of corn ßour, wheat germ ßour, yeast powder, agar, sugar, sorbic acid, vitamin C, linoleic acid, and Þlter paper. After emergence, the adults were fed an artiÞcial diet consisting of yeast powder, honey, sugar, vitamin C, and water in rearing boxes in the laboratory. The whole life stages of B. dorsalis were maintained in a temperature-controlled room at 27 ⫾ 1⬚C, 70 ⫾ 5% relative humidity (RH) with a photoperiod of 14:10 (L:D) h. The 3- to 5-d-old adults in these colonies were used for the toxicity assays and biochemical studies. Voucher specimens were deposited at the insect collection of Southwest University, Chongqing, China. Chemicals. Nine insecticides including malathion (89.1%), chlorpyrifos (95.0%), phoxim (87.8%), triazophos (88.0%), ␤-cypermethrin (95.0%), deltamethrin (99.0%), abamectin (93.0%), imidacloprid (97.5%), and Þpronil (95.0%), as well as three types of synergists including TPP (97.0%), PBO (97.0%), and

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DEM (98.0%), were used in this study. These chemicals were provided by the Institute for Control of Agrochemicals, Sichuan Province, China. 1-naphthyl acetate was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Eserine (SigmaÐ Aldrich, St. Louis, MO) and other biochemical reagents were analytical grade. Bioassay. The susceptibilities of B. dorsalis to nine insecticides were evaluated using the micro-drop method. Each insecticide was dissolved and diluted to six different concentrations with acetone. The test insects were anesthetized by exposure to a low temperature (⫺20⬚C) for no longer than 30 s. Subsequently, a droplet (0.25 ␮l) of insecticide solution was applied topically onto the thoracic tergum of the insects tested with a hand micro-applicator (Hamilton, Reno, NV). The treated insects were reared and fed with the artiÞcial diet in a petri dish (d ⫽ 15 cm) and maintained at 27 ⫾ 1⬚C, 70 ⫾ 5% RH with a photoperiod of 14:10 (L:D) h. Approximately 30 ßies were used for each replicate, and three replicates were tested for each insecticide treatment. Controls were treated with acetone alone. Mortality was checked after 24 h. The insects were considered dead if they did not move after stimulation with a camel hairbrush. For synergism experiments, the synergist (TPP, PBO, and DEM) was mixed with different insecticides (3:1 in volume) and dissolved in acetone. The mixtures were diluted to six different concentrations with acetone. The test insects were treated with 0.25 ␮l of the insecticide and synergist mixture that was applied by a hand micro-applicator as mentioned above. Determination of CarEs Activity. According to the bioassay results, we applied the amount of insecticide corresponding to the LD20 of ␤-cypermethrin, ␤-cypermethrin ⫹ TPP, ␤-cypermethrin ⫹ DEM, or ␤-cypermethrin ⫹ PBO, to the KM population of B. dorsalis. We collected the insects from different treatments as described above at 2, 4, 8, 12, and 24 h for use in enzyme assays. The whole body of the treated insects was examined, along with the dissected midgut, fat body, and Malpighian tubules from insects after 24 h of induction with the various ␤-cypermethrin and synergist treatments. Noninduced insects (without insecticide or synergist application) were used as the control group. Twelve adults as well as the midgut, fat body, and Malpighian tubules from 75 adults were sampled for three replications in different treatments. The adults and tissues were ground with liquid nitrogen and homogenized in 2 ml sodium phosphate buffer (40 mM, pH 7.0). The homogenates were centrifuged at 10,000 ⫻ g at 4⬚C for 10 min. Subsequently, the supernatant was recentrifuged at 10,000 ⫻ g for 5 min. The resulting supernatant was collected and used as a source of enzyme for CarEs activity assays. Protein contents of the enzyme samples, as internal controls to quantify the amount of the sample, were determined following the method of Bradford (1976) using bovine serum albumin (PBS) as a standard. CarEs activity was analyzed with the method described by Van Aspern

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Table 1.

Toxicity of nine insecticides against two populations of B. dorsalis

Classes Organophosphorus

Insecticides Malathion Chlorpyrifos Phoxim Triazophos

Pyrethroids

␤-cypermethrin Deltamethrin

Biopesticide

Abamectin

Neonicotinoid

Imidacloprid

Phenyl pyrazole derivatives

Fipronil

a b c

Vol. 106, no. 2

Populationa

Slope ⫾ SEb

LD50 (ng/ßy) (95% CI)

␹2c

KM DG KM DG KM DG KM DG KM DG KM DG KM DG KM DG KM DG

3.85 ⫾ 0.41 3.36 ⫾ 0.35 2.54 ⫾ 0.28 3.10 ⫾ 0.22 2.08 ⫾ 0.16 2.90 ⫾ 0.24 6.59 ⫾ 0.74 3.10 ⫾ 0.22 1.89 ⫾ 0.15 2.19 ⫾ 0.20 1.75 ⫾ 0.20 1.31 ⫾ 0.10 1.95 ⫾ 0.19 1.50 ⫾ 0.16 1.31 ⫾ 0.11 1.20 ⫾ 0.14 4.47 ⫾ 0.51 4.31 ⫾ 0.42

73.30 (66.60Ð78.00) 230.10 (211.70Ð249.30) 28.38 (18.96Ð37.92) 19.68 (18.06Ð21.26) 6.99 (4.60Ð9.64) 13.65 (8.53Ð22.14) 16.05 (15.16Ð16.76) 11.66 (9.73Ð13.55) 25.50 (22.40Ð29.00) 123.10 (110.00Ð135.90) 33.17 (27.82Ð38.62) 30.44 (25.36Ð35.96) 11.80 (9.00Ð13.60) 23.70 (18.60Ð8.90) 187.48 (52.23Ð223.75) 439.11 (362.50Ð552.45) 1.55 (1.23Ð1.63) 2.07 (1.82Ð2.35)

0.77 2.02 7.73 4.72 6.69 8.96 0.77 7.25 1.56 0.91 1.61 2.58 1.23 2.32 6.27 6.65 5.57 7.15

DG and KM refer to populations from Dongguan of the Guangdong Province and Kunming of the Yunnan Province, China, respectively. Each value represents the mean of three independent measurements. ␹2 goodness-of-Þt test.

(1962) with minor modiÞcations. In brief, 125 ␮l of substrate solution (mixed with 1 ml of 0.03 mol/L 1-naphthylacetate, 1 ml of 10⫺4 mol/L serine, and 98 ml of 0.04 mol/L phosphate buffer, pH 7.0) and 25 ␮l of enzyme source solution were mixed in the well of a 96-well microplate. After incubation for 10 min at 30⬚C, 25 ␮l of the conjugate dye (5% wt:vol sodium dodecyl sulfate 1% wt:vol fast blue B salt ⫽ 5:2 V/V) was added. After a second incubation for 10 min at 30⬚C, the absorbance was determined at a wavelength of 600 nm with a X-Mark microplate absorbance spectrophotometer (Bio-Rad, Hercules, CA). PBS instead of the enzyme supernatant served as a negative control. The standard curve was constructed using different concentrations of 1-naphthol (total volume was 175 ␮l) with 25 ␮l of the fast blue conjugate dye. Subsequently, the enzyme activity was calculated from the 1-naphthol standard curve. From the standard curve, speciÞc activity data are presented as ␮mol/␮g/10 min. Statistical Analysis. Mortality data were corrected with AbbottÕs formula (Abbott 1925) and analyzed by probit analysis to determine the median lethal dosage (LD50). The 95% CI for each LD50 value were also determined using the same procedures. The ␹2 value was used to measure the goodness-of-Þt of the probit regression equation. An unsynergized populationÕs response to a particular insecticide was considered signiÞcantly different from the synergized populationÕs response if their LD50 CIs at 95% did not overlap (Shelton et al. 1993). Synergism ratios (SR) were calculated as follows: SR ⫽ LD50 value of insecticide alone/LD50 value of insecticide with synergist. Enzyme data were analyzed using the analysis of variance (ANOVA), and the means were separated by Duncan multiple range test for signiÞcance (P ⫽ 0.05) using SPSS (version 16.0, SPSS, Chicago, IL).

Results Bioassay. The toxicities of the nine insecticides against the two populations (KM and DG) of B. dorsalis were compared using the micro-drop method. For the KM population, the insects were most sensitive to Þpronil, followed by phoxim, abamectin, triazophos, ␤-cypermethrin, chlorpyrifos, deltamethrin, malathion, and imidacloprid, with LD50 values of 1.55, 6.99, 11.80, 16.05, 25.50, 28.38, 33.17, 73.30, and 187.48 ng/ßy, respectively (Table 1). For the DG population, Þpronil was also the most toxic compound with an LD50 value of 2.07 ng/ßy, followed by triazophos, phoxim, chlorpyrifos, abamectin, deltamethrin, ␤-cypermethrin, malathion, and imidacloprid, with LD50 values of 11.66, 13.65, 19.68, 23.70, 30.44, 123.10, 230.10, and 439.11 ng/ßy, respectively (Table 1). Considering the two populations together, Þpronil was the most toxic, and imidacloprid was the least toxic compound against B. dorsalis. The nonoverlapping 95% CI of LD50 values of malathion, ␤-cypermethrin, abamectin, imidacloprid, and Þpronil between the two populations suggested that there was a statistically signiÞcant difference in the toxicity of these Þve insecticides between the KM and DG populations. However, the other four insecticides did not show signiÞcantly different toxicity between the two populations. Synergists. The synergism of PBO, TPP, and DEM toward four organophosphorus insecticides against two populations of B. dorsalis is shown in Table 2. The results showed that the addition of PBO, TPP, and DEM signiÞcantly increased the toxicity of triazophos against the KM population with a synergism ratio of 2.50-, 3.75-, and 2.54-fold, respectively, and the synergism was signiÞcantly different between PBO and TPP. However, the three synergists did not exhibit a similarly synergistic effect to triazophos against the DG population (SR ⫽ 1.49, 1.87, and 1.71, respectively). For malathion, the three synergists had a

April 2013 Table 2.

WANG ET AL.: SUSCEPTIBILITY OF B. dorsalis TO INSECTICIDES

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Synergism effect of PBO, DEM, and TPP on four organophosphorus insecticides against two populations of B. dorsalis

Insecticides (⫹synergist) Malathion (⫹PBO) (⫹TPP) (⫹DEM) Chlorpyrifos (⫹PBO) (⫹TPP) (⫹DEM) Phoxim (⫹PBO) (⫹TPP) (⫹DEM) Triazophos (⫹PBO) (⫹TPP) (⫹DEM)

Populationa

Slope ⫾ SEb

LD50 (ng/ßy) (95% CI)

␹2c

SRd

KM DG KM DG KM DG

2.73 ⫾ 0.41 2.61 ⫾ 0.21 2.98 ⫾ 0.44 2.35 ⫾ 0.17 2.37 ⫾ 0.40 2.19 ⫾ 0.14

61.08 (51.57Ð76.68) 84.43 (69.94Ð104.46) 67.87 (56.84Ð83.00) 90.19 (67.97Ð114.55) 38.78 (27.89Ð48.93) 88.38 (73.19Ð106.27)

2.58 7.77 0.90 6.04 2.78 7.43

1.20 2.73 1.08 2.55 1.89 2.60

KM DG KM DG KM DG

3.13 ⫾ 0.23 3.25 ⫾ 0.22 2.91 ⫾ 0.25 2.62 ⫾ 0.20 3.70 ⫾ 0.32 2.69 ⫾ 0.21

10.75 (9.96Ð11.59) 13.80 (12.70Ð14.92) 11.54 (10.35Ð12.66) 14.77 (10.87Ð19.24) 30.32 (27.74Ð32.66) 15.98 (14.38Ð17.70)

1.44 3.65 1.36 7.01 2.97 0.57

2.64 1.43 2.46 1.33 0.94 1.23

KM DG KM DG KM DG

3.07 ⫾ 0.23 4.39 ⫾ 0.41 3.26 ⫾ 0.27 4.13 ⫾ 0.44 2.74 ⫾ 0.22 2.77 ⫾ 0.27

7.58 (6.91Ð8.27) 13.86 (10.51Ð18.84) 5.72 (4.37Ð7.15) 10.73 (9.94Ð11.46) 6.17 (4.63Ð7.90) 12.47 (9.93Ð16.39)

0.28 13.93 6.72 1.51 5.91 5.49

0.92 0.98 1.22 1.27 1.13 1.09

KM DG KM DG KM DG

3.45 ⫾ 0.26 4.34 ⫾ 0.39 3.29 ⫾ 0.27 3.39 ⫾ 0.25 3.85 ⫾ 0.31 2.49 ⫾ 0.26

6.42 (5.96Ð6.93) 7.81 (7.23Ð8.34) 4.28 (3.89Ð4.65) 6.22 (5.23Ð7.20) 6.32 (4.87Ð7.85) 6.82 (4.87Ð9.49)

1.82 1.72 4.74 7.00 8.71 7.77

2.50 1.49 3.75 1.87 2.54 1.71

a

DG and KM refer to populations from Dongguan of the Guangdong Province and Kunming of the Yunnan Province, China, respectively. Each value represents the mean of three independent measurements. ␹2 goodness-of-Þt test. d Synergism ratio. b c

stronger effect on the DG population (SR ⫽ 2.55Ð2.73) compared with the KM population (SR ⫽ 1.2Ð1.89). However, there were no signiÞcant differences among the three synergists. PBO and TPP had signiÞcant synergistic effects to chlorpyrifos toxicities against the KM population, with synergism ratios of 2.64 and 2.46, respectively, but they showed a small synergistic effect against the DG population. When applied with phoxim, the three synergists all showed Table 3.

Synergism effect of PBO, DEM, and TPP on two pyrethroids against two populations of B. dorsalis

Insecticides (⫹synergist)

␤-cypermethrin (⫹PBO) (⫹TPP) (⫹DEM) Deltamethrin (⫹PBO) (⫹TPP) (⫹DEM)

a

no signiÞcantly synergistic effects against the two populations. The analysis with pyrethroid insecticides suggested that the three synergists could signiÞcantly increase toxicity of both ␤-cypermethrin and deltamethrin, with a higher extent against the DG population compared with that of the KM population (Table 3). PBO and TPP had better synergistic effects than DEM toward ␤-cypermethrin against the two populations.

Populationa

Slope ⫾ SEb

LD50 (ng/ßy) (95% CI)

␹2c

SRd

KM DG KM DG KM DG

0.52 ⫾ 0.11 2.11 ⫾ 0.21 1.46 ⫾ 0.42 2.93 ⫾ 0.25 1.63 ⫾ 0.43 1.64 ⫾ 0.17

10.37 (6.28Ð16.08) 27.72 (24.41Ð30.88) 10.67 (6.44Ð14.84) 30.44 (27.96Ð32.89) 12.88 (8.57Ð17.28) 43.34 (23.94Ð62.27)

0.36 4.02 1.89 3.11 1.59 9.98

2.46 4.44 2.39 4.04 1.98 2.84

KM DG KM DG KM DG

1.73 ⫾ 0.15 1.29 ⫾ 0.13 1.63 ⫾ 0.16 1.32 ⫾ 0.11 1.95 ⫾ 0.16 0.96 ⫾ 0.11

30.84 (22.06Ð40.60) 7.25 (5.69Ð8.92) 28.79 (24.17Ð33.53) 14.97 (12.35Ð18.07) 24.43 (17.42Ð31.54) 13.59 (9.50Ð17.83)

7.60 1.42 4.90 4.13 7.47 3.07

1.08 4.20 1.15 2.03 1.36 2.24

DG and KM refer to populations from Dongguan of the Guangdong Province and Kunming of the Yunnan Province, China, respectively. Each value represents the mean of three independent measurements. ␹2 goodness-of-Þt test. d Synergism ratio. b c

974 Table 4. Insecticides (⫹synergist) Abamectin (⫹PBO) (⫹TPP) (⫹DEM)

JOURNAL OF ECONOMIC ENTOMOLOGY

Vol. 106, no. 2

Synergism effect of PBO, DEM, and TPP on abamectin against two populations of B. dorsalis Populationa

Slope ⫾ SEb

LD50 (ng/ßy) (95% CI)

␹2c

SRd

KM DG KM DG KM DG

1.30 ⫾ 0.19 1.80 ⫾ 0.17 1.27 ⫾ 0.24 2.15 ⫾ 0.18 1.22 ⫾ 0.18 1.41 ⫾ 0.15

5.26 (4.04Ð6.40) 5.29 (4.40Ð6.15) 5.53 (4.29Ð6.80) 6.67 (5.84Ð8.58) 9.29 (7.66Ð11.56) 7.22 (6.08Ð8.72)

2.16 3.76 1.77 2.84 0.98 4.82

2.24 4.48 2.13 3.55 1.27 3.28

a

DG and KM refer to populations from Dongguan of the Guangdong Province and Kunming of the Yunnan Province, China, respectively. Each value represents the mean of three independent measurements. ␹2 goodness-of-Þt test. d Synergism ratio. b c

The toxicity of abamectin against the DG population was signiÞcantly increased by adding PBO, TPP, and DEM with SRs of 4.48, 3.55, and 3.28, respectively. In the KM population, it was signiÞcantly enhanced by PBO (SR ⫽ 2.24) and TPP (SR ⫽ 2.13) but was not signiÞcantly affected by DEM (SR ⫽ 1.27) (Table 4). Assays of imidacloprid with synergists demonstrated that DEM had a signiÞcantly synergistic effect toward imidacloprid against the DG population (SR ⫽ 2.57). PBO and DEM exhibited little synergistic effects in the two populations. Conversely, the effect of imidacloprid against the KM population was unchanged by adding DEM (SR ⫽ 0.85) (Table 5). The results from the experiment of Þpronil with synergists indicated that after 24 h of exposure, the synergists had no signiÞcantly synergistic effects for Þpronil, excluding DEM to the KM population (Table 6). Activity of CarEs. We conducted CarEs activity assays in the KM population after the addition of ␤-cypermethrin and ␤-cypermethrin-synergist mixtures at sublethal doses (corresponding to the LD20 from the bioassays). The speciÞc activity of CarEs in the KM population of B. dorsalis with different treatments all showed a tendency to increase at 4 Ð 8 h and then declined within 24 h (Fig. 1). The activities of CarEs after treatment with ␤-cypermethrin by adding PBO, TPP, or DEM were lower compared with that of the ␤-cypermethrin alone treatment. The speciÞc activity of CarEs was signiÞcantly higher in all of these tissues than in the whole body of Table 5. Insecticides (⫹synergist) Imidacloprid (⫹PBO) (⫹TPP) (⫹DEM)

a

B. dorsalis (Fig. 2). In most cases, the CarEs activity within the fat body was higher compared with those in the midgut and Malpighian tubules. In general, the addition of each synergist lowered CarEs activity in comparison to the ␤-cypermethrin alone treatment. PBO led to a strong decrease in CarEs activity in all three tissues tested. Discussion B. dorsalis has become a major insect pest in various orchard crops. As a polyphagous species, this insect has the potential to invade new areas and adapt to new host plants (Clarke et al. 2005). There is a long history of using organophosphates in fruit production. The present work has conÞrmed that the LD50 values for malathion treatment in both populations was as high as 73.30 and 230.10 ng/ßy, whereas the LD50 was 44 ng/ßy in 2004 (Hsu et al. 2004). For ␤-cypermethrin, the lowest toxic dose in 2004 was 5.0 ng/ßy. However, in our study, the lowest toxic dose was 25.50 ng/ßy. These data possibly suggest that the resistance of B. dorsalis to malathion and pyrethroids has increased rapidly in recent years. It has also been reported that B. dorsalis has expressed high resistance to trichlorfon and ␤-cypermethrin in different populations (Jin et al. 2011); no evidence has been shown that B. dorsalis has developed resistance to chlorpyrifos, phoxim, and triazophos. Our study conÞrmed that B. dorsalis was sensitive to these three insecticides at the time of testing. We found that B. dorsalis was most sensitive to

Synergism effect of PBO, DEM, and TPP on imidacloprid against two populations of B. dorsalis Populationa

Slope ⫾ SEb

LD50 (ng/ßy) (95% CI)

␹2c

SRd

KM DG KM DG KM DG

2.01 ⫾ 0.14 1.48 ⫾ 0.13 1.55 ⫾ 0.17 1.73 ⫾ 0.17 1.53 ⫾ 0.14 1.92 ⫾ 0.18

103.60 (90.78Ð117.91) 406.89 (344.61Ð506.17) 109.36 (86.26Ð130.68) 274.95 (235.66Ð316.84) 221.83 (186.98Ð261.58) 171.14 (144.61Ð198.11)

5.24 0.78 5.63 2.73 6.70 2.28

1.81 1.08 1.71 1.60 0.85 2.57

DG and KM refer to populations from Dongguan of the Guangdong Province and Kunming of the Yunnan Province, China, respectively. Each value represents the mean of three independent measurements. ␹2 goodness-of-Þt test. d Synergism ratio. b c

April 2013 Table 6. Insecticides (⫹synergist) Fipronil (⫹PBO) (⫹TPP) (⫹DEM)

WANG ET AL.: SUSCEPTIBILITY OF B. dorsalis TO INSECTICIDES

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Synergism effect of PBO, DEM, and TPP on fipronil against two populations of B. dorsalis Populationa

Slope ⫾ SEb

LD50 (ng/ßy) (95% CI)

␹2c

SRd

KM DG KM DG KM DG

3.18 ⫾ 0.25 4.16 ⫾ 0.36 3.93 ⫾ 0.35 4.63 ⫾ 0.39 2.48 ⫾ 0.20 3.72 ⫾ 0.33

1.24 (1.133Ð1.34) 1.77 (1.63Ð1.89) 1.22 (1.114Ð1.31) 1.97 (1.53Ð2.49) 0.77 (0.546Ð1.01) 1.69 (1.15Ð2.17)

1.91 2.14 4.86 12.52 6.41 11.97

1.25 1.17 1.27 1.05 2.01 1.22

a

DG and KM refer to populations from Dongguan of the Guangdong Province and Kunming of the Yunnan Province, China, respectively. Each value represents the mean of three independent measurements. ␹2 goodness-of-Þt test. d Synergism ratio. b c

Þpronil; the LD50 values were noticeably lower in the KM and DG populations, and were 1.55 and 2.07 ng/ ßy, respectively. Fipronil is a phenylpyrazole derivative and systemic insecticide that has been used extensively worldwide for controlling broad spectrum and sucking insects, and it has been reported that Þpronil has high efÞcacy in pest control even at a low Þeld application rate (Aajoud et al. 2003). Based on similar compounds to those tested here, these results may guide the development and testing of new insecticides for B. dorsalis management. Moreover, avermectin is an antibiotic insecticide, which has low mammal toxicity and high toxicity to B. dorsalis. Therefore, avermectin remains a good choice for fruit protection in orchards and Þelds. The LD50 of malathion, ␤-cypermethrin, abamectin, imidacloprid, and Þpronil were signiÞcantly different between the two populations. This result conÞrms previous studies that show insecticide sensitivity depends on many factors including environmental and Þeld conditions, and the history of pesticide exposure to that particular insect population.

Synergists, which block certain insecticide metabolic enzymes, can be used to determine the potential mechanisms involved in tolerance variation. The aim of the current study was to identify the synergistic effects of three synergists toward nine insecticides in B. dorsalis. The study results suggested that the malathion resistance of this pest in the DG population and triazophos resistance in the KM population were all associated with enhanced activity of P450s, GSTs, and CarEs. P450s and CarEs were the most important enzymes in chlorpyrifos detoxiÞcation in the KM population. From the synergistic results, we could not determine which type of enzyme played an important role in enhanced phoxim detoxiÞcation, and these synergists had no signiÞcant effect on malathion in the KM population, which suggests that other resistance mechanisms might be involved. Overall, these data were all in agreement with previous works in which researchers have found that enhanced activity of metabolic detoxiÞcation is a major mechanism for organophosphate resistance. For example, malathion resistance of a Locusta migratoria manilensis (Meyen)

Fig. 1. Effects of ␤-cypermethrin (B), ⫹ TPP (B ⫹ T), ⫹ DEM (B ⫹ D), and ⫹ PBO (B ⫹ P) at an LD20 dose on speciÞc activities of CarEs in B. dorsalis. Each value represents the mean ⫾ SE of three independent experiments.

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Fig. 2. Effects of ␤-cypermethrin (B), ⫹ TPP (B ⫹ T), ⫹ DEM (B ⫹ D), and ⫹ PBO (B ⫹ P) at an LD20 dose on speciÞc activities of CarEs in different tissues of B. dorsalis. Each value represents the mean ⫾ SE of three independent experiments. A, B, and C above each bar indicate a signiÞcant difference among different tissues and a, b, and c above each bar indicate a signiÞcant difference among different treatments by ANOVA followed by the DuncanÕs multiple range test (P ⬍ 0.05).

Þeld-resistant population was conferred by multiple mechanisms, including increased detoxiÞcation by CarEs and GSTs, but P450s were unlikely to be involved in the resistance of chlorpyrifos and phoxim (Yang et al. 2009). It has been reported that the resistance mechanisms of B. dorsalis to naled, trichlorfon, and malathion might be caused by esterases (Hsu et al. 2004). Resistance of Lucilia cuprina (Wiedmann) and Nilaparvata lugens (Stål) to organophosphorus insecticides was because of esterase gene mutations and high esterase activity (Heidari et al. 2005, Latif et al. 2010). However, based on the present results, we found that GSTs, P450s, and CarEs might all be involved in detoxiÞcation of organophosphorus insecticides in B. dorsalis. Our results demonstrated that the exact detoxiÞcation mechanisms involved were strongly dependent upon the insecticide itself and the insect population, which was in agreement with a previous study of insecticide resistance in Culex quinquefasciatus Say (Gordon and Ottea 2012). Comparing the two different populations, we found that the three synergists played an important role in triazophos and chlorpyrifos resistance in the KM population but were the most important in malathion resistance and detoxiÞcation in the DG population. These results demonstrate that the three enzymes appear to have different important roles in different populations, which agrees with the bioassay results. Our results also suggested that both P450s and CarEs were involved in abamectin resistance in the two populations. Similar results were found in the study of Plutella xylostella (L.), which indicated that mixed function oxidase enzymes were the most important enzymes in abamectin detoxiÞcation and resistance. GSTs and esterase may play some roles, especially in an abamectin resistant strain (Qian et al. 2008).

It has been reported that quantitative and qualitative changes in CarEs might contribute together to cause pyrethroid resistance in a resistant strain of houseßy (Zhang et al. 2007, 2010). In our study of synergistic effects, we found that P450 mixed function oxidase systems were not the only mechanism of resistance in this pest; GSTs and CarEs may also be involved in ␤-cypermethrin detoxiÞcation, especially P450s and CarEs, because relatively high PBO and TPP synergistic effects occurred in the two populations. Studies have also demonstrated that pyrethroid resistance in Spodoptera litura (F.) is associated with the enhanced activities of P450s and esterase (Huang and Han 2007). In our CarEs activity study, we found that not only GSTs but also P450s and CarEs are involved in ␤-cypermethrin detoxiÞcation. Pyrethroid resistance, owing to an increased level of GSTs, has been detected in N. lugens (Vontas et al. 2001), Bovicola ovis (Schrank) (Jazayeri 2004), and Aedes aegypti (L.) (Jagadeshwaran and Vijayan 2009). Our synergism and CarEs activity experiment showed that to a greater or lesser extent GSTs, P450s, and CarEs were all involved in detoxiÞcation of pyrethroid. In our previous studies, we puriÞed GSTs and AChE from four Þeld populations of B. dorsalis, and found that the activities were different among the four populations (Hu et al. 2011, Shen et al. 2012). Although we have not investigated the characteristics of P450s and CarEs in different populations, we speculate that there are differences in the activity levels of P450s and CarEs among the different populations, and this may be why the same synergist and insecticide mixture can have a different synergistic ratio in different populations. GSTs might play an important role in imidacloprid resistance in the DG population, but our study revealed little regarding the resistance mechanisms of B. dorsalis to this pesticide in the KM population. From

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the results of the synergistic experiment, the resistance mechanism of B. dorsalis to Þpronil remained unclear. Imidacloprid is a neonicotinoid insecticide that affects nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity (Matsuda et al. 2001, Liu et al. 2005), and Þpronil is known to inhibit GABA receptors in insects (Cole et al. 1993). This may explain why PBO, DEM, and TPP showed little effectiveness with these two insecticides. We found that insecticide and synergist treatments led to dynamic changes in the CarEs activities within 24 h. This suggests that to adapt and restore external environmental stimuli, B. dorsalis has major changes in the interior of their bodies, which in turn affected the abundance of CarEs and its activity. When we compared CarEs speciÞc activity among different tissues, we found that higher esterase activity were expressed in the fat body, midgut, and Malpighian tubules compared with that in the whole body. This suggests that these tissues, and in particular the fat body, are important tissues for detoxiÞcation. Compared with the control group, addition of sublethal dosage of ␤-cypermethrin induced CarEs activity in the three tissues tested. The results suggest that CarEs may play a role in ␤-cypermethrin detoxiÞcation and play a role in resistance to pesticide exposure. Given the evidence from Helicoverpa armigera (Hu¨ bner), researchers have found several-fold more transcripts of the four esterase genes in the fat body of the resistant strain compared with that of the susceptible strain, and for CCE001a, the gene levels were twofold higher in the midgut and 90-fold higher in the fat body (Wu et al. 2011). When applied with ␤-cypermethrin, TPP signiÞcantly inhibited CarEs activity in the three tissues, which further suggests that CarEs might mediate the resistance of B. dorsalis. It is known that DEM is an inhibitor of both oxidases and esterases (Eto 1974). DEM is an inhibitor of glutathione S-transferases, and PBO is an inhibitor of mixed function oxidases (Wilkinson 1976). Moreover, we found that the addition of each synergist in combination with a pesticide lowered CarEs activity compared with ␤-cypermethrin alone treatment. These results suggest that CarEs was the major enzyme involved in ␤-cypermethrin detoxiÞcation in B. dorsalis. This observation deserves further investigation. We realize that this study did not include a reference susceptible population in the bioassay, and we rank the relative potency based on LD50 values. Based on this approach, determining the actual resistance levels is difÞcult. However, the results of this work clarify some susceptibilities and primary mechanisms for the different insecticide classes, provide some guidance for the application of insecticides for the control of B. dorsalis, and offer guidance regarding the best synergists to control the losses caused by this insect and slow resistance development. Further studies of these populations are required to clarify resistance mechanisms and provide solutions for sustainable control.

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Acknowledgments This research was funded in part by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0976), the National Basic Research Program of China (2009CB125903 and 2009CB119200), the Natural Science Foundation of Chongqing (CSTC, 2009BA1042), the Earmarked Fund for Agricultural Research System of China (CARS-27-06B), and the Specialized Research Fund for the Doctoral Program of Higher Education in China (20100182120022).

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